摘要
本研究采用PCR技术扩增Fms样酪氨酸激酶-3(fms-like tyrosine kinase 3,FLT3)基因外显子14-15和核仁磷酸蛋白1 (nucleophosmin family, member 1,NPM1)基因外显子11。通过琼脂糖凝胶电泳、变性聚丙烯酰氨凝胶电泳(Denaturing polyacrylamide gel electrophoresis, Denaturing PAGE)与毛细管电泳(Capillary electrophoresis, CE)3种方法对99例初诊急性髓系白血病(acute myeloid leukemia,AML)患者FLT3基因中小片段插入突变(length mutation, FLT3-LM)进行检测；并用变性PAGE和毛细管电泳两种方法对上述患者NPM1基因插入突变进行检测；同时我们还应用PCR-变性PAGE法对44例骨髓增生异常综合征(myelodysplastic syndromes, MDS)患者的FLT3-LM和NPM1插入突变进行检测。采用G显带方法分析了72例AML患者和39例MDS患者的染色体核型。本研究目的是确定FLT3-LM与NPM1突变在初诊AML患者中的发生率、在FAB各亚型患者中的分布、与AML患者染色体核型以及临床疗效之间的关系；通过对部分FLT3或NPM1基因插入突变阳性患者的PCR产物的直接测序,确定该两种基因突变的特点；建立一种检测FLT3-LM与NPM1插入突变灵敏可靠的方法。
结果：
1.在AML与MDS患者中均可检出大量染色体核型异常,AML患者中染色体核型异常率68.06%(49/72)高于MDS患者染色体核型异常率46.15%(18/39)(p<0.05)。
2.AML患者FLT3-LM发生率为30.3%(30/99)。各种电泳技术检测的效率略有不同：琼脂糖凝胶电泳、变性]PAGE、毛细管电泳3种方法的检出率分别为20.2%(20/99),29.29%(29/99),30.3%(30/99)。对照组中MDS患者应用变性PAGE方法的检出率为4.55%(2/44),正常人中未检出。
3.AML患者NPM1基因插入突变发生率为15.15%(15/99)。变性PAGE、毛细管电泳两种方法的检出率分别为11.11%(11/99)和15.15%(15/99),同时检测出2例内含子缺失突变。对照组中MDS患者与正常人应用变性PAGE方法均末检出突变。
4.30例FLT3-LM+的AML患者中26例患者突变型与野生型等位基因比值(allelic ratios, AR)大于0.02,4例患者AR小于0.02。FLT3-LM+ M6型患者中,AR均小于0.05。30例FLT3-LM+患者中28例患者(93.33%)突变条带为1条,2例患者(6.67%)突变条带多于1条。插入突变序列长度3-144bp不等,不同亚型的插入片段有差别。
5.13例FLT3-LM+患者直接测序结果显示,4例插入碱基序列为FLT3野生型片段的完全重复,2例为完全陌生的碱基插入,7例为部分碱基序列重复。所有重复或插入序列发生在p.E573位至p.P606位之间,多集中于p.F590-p.R595之间。
6.10例NPM1基因插入突变阳性患者PCR产物直接测序结果显示,10例均为A型突变(c.860_863dupTCTG)。突变导致NPM蛋白羧基末端读码框移,末尾7个氨基酸WQWRKSL被11个氨基酸CLAVEEVSLRK所代替。2例缺失突变直接测序结果显示,1例为IVS10-18_-15delCTTT,另外1例IVS10-17_-15delTTT.该2例患者为新发现的内含子缺失突变。12例患者中有4例存在3’UTR区缺失1个碱基T(*165delT,rs34351976)的多态性改变。
7.49例核型异常AML患者中FLT3-LM发生率为24.49%(12/49),23例核型正常AML患者中FLT3-LM为43.48%(10/23)(p>0.05)；49例核型异常AML患者中NPM1突变为4.08%(2/49),23例核型正常AML患者中NPM1突变为26.09%(6/23)(p<0.05)。
8.99例AML患者中79例有治疗记录,6例早期死亡(生存期小于14天),可供疗效评价的患者为73例。FLT3-LM+患者中完全缓解(CR)率为36.36%(8/22),低于FLT3-LM患者CR率62.75%(32/51)(p<0.05)。早期死亡6例患者中FLT3-LM+占50%(3/6)。73例可评价疗效患者中,CR患者中FLT3-LM+占20%(8/40)、部分缓解(PR)患者中为38.1%(8/21)、未缓解(NR)患者中为50%(6/12)。49例非M3型患者中FLT3-LM+17例,FLT3-LM32例,达到CR的患者共17例,其中FLT3-LM患者CR率为43.75%(13/32),FLT3-LM+患者CR率17.65%(4/17)(p>0.05)。
9.15例NPM1阳性患者中FLT3-LM发生率为40%(6/15),高于NPM1阴性患者FLT3-LM发生率28.57%(24/84),但无统计学差异(p>0.05)。
10.FLT3-LM-/NPMl+患者的CR率为80%(4/5),FLT3-LM-/NPM1患者的CR率为60.87%(28/46),FLT3-LM+/NPM1患者的CR率为41.18%(7/17)FLT3-LM+/NPM1+患者CR率为20%(1/5)。非M3亚型AML患者的治疗结果为：FLT3-LM-/NPM1+患者CR率为80%(4/5),FLT3-LM-/NPM1患者CR率为37.04%(10/27),FLT3-LM+/NPM 1患者CR率为16.67%(2/12),FLT3-LM+/NPM1+患者CR率为20%(1/5)。
11.在17例FLT3-LM+患者的63份标本中,复查时仍有11例患者为阳性(共20份标本)。出现的阳性条带均与初诊时阳性条带大小相同。
结论：
1.AML与MDS患者中存在大量染色体核型异常,对诊断预后具有重要意义。
2.在FLT3-LM基因突变检测中,相对于琼脂糖凝胶电泳,变性PAGE和毛细管电泳具有较高的分辨率和灵敏度。
3.FLT3-LM与NPM1突变为AML患者中常见基因改变。NPM1突变与FLT3-LM无相关性。
4.FLT3插入基因序列突变长度范围变动较大,部分导致插入点的氨基酸发生取代,其他为整框插入；突变型/野生型AR范围波动较大。
5.部分FLT3-LM+患者插入序列为陌生碱基,FLT3基因突变位置相对较集中,所有重复或插入发生在p.E573位至p.P606位之间,多集中于p.F590-p.R595之间。
6.AML患者中正常核型患者FLT3-LM发生率与异常核型患者无统计学差异,正常核型患者NPM1基因突变发生率高于异常核型患者。
7.NPM1插入突变均为A型突变,突变导致NPM蛋白羧基末端读码框移,末尾7个氨基酸WQWRKSL被11个氨基酸CLAVEEVSLRK所代替。首次在NPM1基因内含子区发现2例缺失突变,突变类型分别为IVS10-18_-15delCTTT和IVS10-17-15delTTT。
8.FLT3-LM对CR率有不利影响,NPM1突变单独发生的患者有较高的CR率,NPM1和FLT3-LM同时发生突变的患者CR率较低,预后不好。
9.FLT3-LM或可作为AML患者微小残留病检测标记。
The aim of this study was to analyze the frequencies of fms-like tyrosine kinase-3(FLT3) and nucleophosmin family, member 1 (NPM1) gene mutations in de novo acute myeloid leukemia(AML) patients, to investigate the relationship between FLT3 length mutation (FLT3-LM) or NPM1 mutations and chromosome alterations, FAB subgroups, as well as efficacy of therapy. We also studied the mutant features of FLT3 and NPMl and evaluated the sensitivibility and reliability of the procedure developed for identifying both FLT3 and NPM1 mutations. Genomic DNA was amplified by PCR. The PCR products were seperated with 2% agarose gel electrophoresis,8% denaturing PAGE or capillary electrophoresis to detect the length mutations of FLT3 gene in 99 de novo AML patients.8% denaturing PAGE and capillary electrophoresis were used to detect the mutations of NPM1 gene. Denaturing PAGE was also used to detect the mutations of FLT3 gene and NPM 1 gene in 44 myelodysplastic syndromes (MDS) patients as controls. Karyotyping was performed in 72 AML patients and in 39 MDS patients by G banding techniques. Some of the PCR products of FLT3 and/or NPMl positive patients were sequenced.
Results:
1. There were various aberrational karyotypes in AML and MDS patients, and the frequencies of abnormal karyotype in AML(68.06%) were higher than that in MDS(46.15%)(p<0.05).
2. The frequencies of FLT3-LM were identified in 30.3%(30/99) of AML patients. The detection rates were different with the electrophoresis techniques employed, 20.2%(20/99) with agarose gel electrophoresis,29.9%(29/99) with denaturing PAGE, and 30.3%(30/99) with capillary electrophoresis, respectively. In control group, 4.55%(2/44) was detected by denaturing PAGE in MDS patients, and none was detected in normal subjects.
3. The frequencies of NPM1 mutations were detected in 15.15%(15/99) of AML patients,11.11%(11/99) with denaturing PAGE, and 15.15%(15/99) with capillary electrophoresis. We also identified 2 intronic deletion mutations. In control group, neither was detected by denaturing PAGE in MDS patients nore in normal subjects.
4. The ratios of mutant alleles to wild alleles of FLT3 allele(AR) in 26 patients were above 0.02, with 4 patients below 0.02. In FLT3-LM+ M6 patients, all of their AR were below 0.05. Twenty eight patients(93.33%) had a single duplicated allele, while there were 2 patients had 2 or more duplicated alleles. The length of inserted sequeces ranged from 3bp to 144bp in all FLT3-LM+ patients.
5. DNA sequences analysis of FLT3-LM+ were performed in 13 patients. Four patients were of pure duplications, and 2 patients had foreign sequences inserted, and the other 7 patients were partial duplications. The duplication or insertion occured in the segment from p.E573 to p.P606 of the FLT3 protein, with the majority clustered in a stretch between codons 590 and 595.
6. DNA sequences analysis of NPM1 mutation were performed on 10 patients, all of them were of A type, which was a 4 bp insertion(c.860_863dupTCTG). The C-terminal portion of the NPM protein by replacing the last seven amino acids(WQWRKSL) with 11 residues(CLAVEEVSLRK). Two new intronic deletion mutations were identified, one case was IVS10-18_-15delCTTT, the other was IVS10-17-15delTTT.
7. In AML patients with abnormal karyotype, the frequencies of FLT3-LM were not different significantly from that in patients with normal karyotype,24.49%(12/49) vs 43.48%(10/23)(p>0.05). While there was a significant difference in frequencies of NPM1 mutation between patients with abnormal karyotype and with normal karyotype 4.08%(2/49) vs 26.09%(6/23) (p<0.05).
8. The complete remission(CR) rate in FLT3-LM+ patients(36.36%) was lower than that in FLT3-LM- patients(62.75%) in the panel of 73 patients(p<0.05). The distributions of FLT3-LM+ were 8 out of 40 in CR patients,8 out of 21 in PR patients, and 6 out of 12 in NR patients. Seventeen patients achieved CR among 49 non-M3 AML patients, of which 17 were FLT3-LM+ and 32 were FLT3-LM-, the CR rate was higher in FLT3-LM- patients (43.75%,14/32) than that in FLT3-LM+ patients (17.65%,3/17) (p>0.05).
9. Fourty percents (6/15) of NPM1+ patients were also FLT3-LM+, which is higher than NPMl-FLT3+ patients(28.57%,24/84), but there was not statistical significance.
10. The CR rate in FLT3-LM-/NPM+ patients was 80%(4/5),60.87%(28/46) in NPMl-/FLT3-LM- patients, and 41.18%(7/17) in FLT3-LM+/NPM1- patients, and 20%(1/5) in FLT3-LM+/NPM1+ patients. In the non-M3 AML patients group, the CR rate was 80%(4/5) with FLT3-LM-/NPM1+, and 37.04%(10/27) with FLT3-LM-/NPM1-,and 16.67%(2/12) with FLT3-LM+/NPM1-, and 20%(1/5) with FLT3-LM+/NPM1+, respectively.
11. Twenty samples from 11 patients remained positive in 63 samples of 17 FLT3-LM+ patients followed-up. The lengths of duplication remained the same as that detected when the initial diagnose was made.
Conclusions:
1. There were plenty of chromosome aberrations in AML and MDS patients, which were important to dignosis and prognosis.
2. Denaturing PAGE and capillary electrophoresis were more sensitive than agarose gel electrophoresis in detecting FLT3 length mutations.
3. FLT3 length mutation and NPM1 mutation were the common genetic evidences in AML patients. There was no correlation between NPM1 and FLT3-LM mutations.
4. The insert sequences of FLT3-LM+ranged from 3bp to 144bp, some insertions led to substitution of amino acids, others remained the reading frame. The ratios of mutant allele to wild allele ranged from 0.01 to 3.
5. Some of the insert sequences of FLT3-LM positive patients were foreign bases. The duplication or insertion occured in the region from p.E573 to p.P606 of the FLT3 protein, with the majority clustered in a stretch between codons 590 and 595.
6. There was no statistics significance in the frequencies of FLT3-LM+ between AML patients with normal karyotype and with abnormal karyotype, while the frequencies of NPM1 mutation were higher in patients with normal karyotype than that in patients with abnormal karyotype.
7. The NPM1 mutations were all of A type, which was a 4 bp insertion (860_863dupTCTG). The C-terminal portion of the NPM protein by replacing the last seven amino acids(WQWRKSL) with 11 residues(CLAVEEVSLRK).Two intronic deletions were novel, one mutation was IVS10-18_-15delCTTT, and the other was IVS10-17-15delTTT.
8. FLT3-LM was associated with lower CR rate. FLT3-LM-/NPM1+ was associated with higher CR rate, while FLT3-LM+/NPM1+ was associated with lower CR rate and bad prognosis.
9. As a following-up marker, FLT3-LM+ could be used to detect the minimal residual disease for AML patients.
引文
1. Grimwade D, Walker H, Oliver F, et al. The importance of diagnostic cytogenetics on outcome in AML:analysis of 1612 patients entered into the MRC AML 10 trial. Blood,1998,92:2322-33.
2. Byrd JC, Mrozek K, Dodge RK, et al. Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia:results from cancer and leukemia Group B(CALGB 8461). Blood,2002,100:4325-36.
3. Slovak ML, Kopecky KJ, Cassileth PA, et al. Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia:a Southwest Oncology Group/Eastern Cooperative Oncology Group study. Blood,2000,96:4075-83.
4. Mrozek K, Heerema NA and Bloomfield CD. Cytogenetics in acute leukemia. Blood reviews,2004,18:115-36.
5. Schlenk RF, Dohner K, Krauter J, et al. Mutations and Treatment Outcome in Cytogenetically Normal Acute Myeloid Leukemia. N Engl J Med,2008,358:1909-18.
6. Ley TJ, Mardis ER, Ding L, et al. DNA sequencing of a cytogenetically normal acute myeloid leukaemia genome. Nature,2008,456:66-72.
7. Small D. Targeting FLT3 for the treatment of leukemia [J]. Semin Hematol,2008, 45(3Suppl 2):S17-S21.
8. Murphy KM, Levis M, Hafez MJ, et al. Detection of FLT3 internal tandem duplication and D835 mutations by a multiplex polymerase chain reaction and capillary electrophoresis assay. J Mol Diagn,2003,5:96-102.
9. Grisendi S, Mecucci C, Falini B, et al. Nucleophosmin and cancer[J]. Nat Rev Cancer, 2006,6(7):493-505.
10. Nakagawa M, Kameoka Y, Suzuki R. Nucleophosmin in acute myelogenous leukemia. N Engl J Med,2005,352:1819-1820.
11. Renneville A, Roumier C, Biggio V, et al. Cooperating gene mutations in acute myeloid leukemia:a review of the literature. Leukemia,2008,22:915-931.
12. Schnittger S, Schoch C, Dugas M, et al. Analysis of FLT3 length mutations in 1003 patients with acute myeloid leukemia:correlation to cytogenetics, FAB subtype, and prognosis in the AMLCG study and usefulness as a marker for the detection of minimal residual disease. Blood,2002,100:59-66.
13.邹积艳,朱平,刘红星等.白血病NPM1基因突变检测方法的临床适用性比较.中华检验医学杂志,2009,32(1)：35-39.
14.张之南,沈悌.白血病诊断及疗效标准[M].第3版.北京：科学出版社,2007
15. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res,1988,16(3):1215.
16. Fey MF, Pilkington SP, Summers C, et al. Molecular diagnosis of haematological disorders using DNA from stored bone marrow slides. Br J Haematol,1987, 67:489-492.
17. ISCN(2005):An international System for Human Cytogenetic Nomenclature, Shaffer LG, Tommerup N. (eds); S. Karger, Basel 2005.
18. Meshinchi S, Woods WG, Stirewalt DL, et al. Prevalence and prognostic significance of FLT3 internal tandem duplication in pediatric acute myeloid leukemia. Blood, 2001,97:89-94.
19. Falini B, Mecucci C, Tiacci E, et al. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N Engl J Med,2005,352:254-266.
20. Quentmeier H, Martelli MP, Dirks WG, et al. Cell line OCI/AML3 bears exon-12 NPM gene mutation-A and cytoplastic expression of nuclephosmin. Leukemia, 2005,19:1760-1767.
21.马亮,钟明华,廖军鲜,等.美国西南肿瘤组/东部肿瘤协作组染色体核型分组法与急性髓系白血病预后关系的研究.白血病.淋巴瘤,2010,19(1)：20-22.
22.马亮,钟明华,李振玲,等.染色体核型分析在骨髓增生异常综合征中的意义.中日友好医院学报,2008,22(3)：145-147
23. Frohling S, Schlenk RF, Breitruck J, et al. Prognostic significance of activating FLT3 mutation in younger adults(16 to 60 years) with acute myeloid leukemia and normal cytogenetics:a study of the AML Study Group Ulm. Blood,2002,100:4372-4380.
24.韩阳利,张苏江,乔纯,等.急性髓系白血病FLT3基因突变研究.中国实验血液学杂志,2009,17(5)：1135-1139.
25. Li J, Wang L, Mamon H, et al. Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing. Nat Med,2008, 14(5):579-84.
26.中美联合上海市白血病协作组.468例急性白血病患者FLT3基因突变及其预后价值分析.中华血液学杂志,2010,31：1-5.
27. Bang SM, Ahn JY, Park J, et al. Low frequency and variability of FLT3 mutations in Korean patients with acute myeloid leukemia. J Korean Med Sci,2008,23:833-837.
28.刘红,于红,贾红英,等.恶性血液病FLT3基因突变的检测及临床意义.中国实验血液学杂志,2007,15(4)：709-713.
29.徐兵,唐家宏,李琳,等.血液肿瘤患者FLT3/ITD基因突变的检测及临床意义.中国实用内科杂志,2007,27(14)：1113-1115.
30. Georigiou G, Karali V, Zouvelou C, et al. Serial determination of FLT3 mutations in myelodysplastic syndrome patients at diagnosis, follow up or acute myeloid leukaemia transformation:incidence and their prognostic significance. Br J Haematol, 2006,134:302-306.
31. Pinheiro RF, Moreira ED, Silva MRR, et al. FLT3 internal tandem duplication during myelodysplastic syndrome follow-up:a marker of transformation to acute myeloid leukemia. Cancer Genet Cytogenet,2008,183:89-93.
32. Falini B, Mecucci C, Tiacci E, et al Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N Engl J Med,2005,352:254-266.
33. Thiede C, Koch S, Creutzig E, et al Prevalence and prognostic impact of NPM1 mutation in 1485 adult patients with acute myeloid leukemia(AML). Blood,2006, 107:4011-4020.
34. Ruan GR, Li JL,Qin YZ, et al. Nuclephosmin mutations in Chinese adults with acute myelogenous leukemia. Ann Hematol,2009,88:159-166.
35. Yan LZ, Chen SN, Liang JY, et al. Analysis of NPM1 gene mutations in Chinese adults with acute myeloid leukemia. Int J Hematol,2007,86:143-146.
36.张悦,张美荣,杨琳,等.急性髓系白血病和骨髓增生异常综合征患者NPM基因突变的研究.中华血液学杂志,2006,27(7)：470-473.
37. Thiede C, Steudel C, Mohr B, et al. Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia:association with FAB subtypes and identification of subgroups with poor prognosis. Blood,2002,99:4326-35.
38. Schnittger S, Kohl TM, Haferlach T, et al. KIT-D816 mutations in AML1-ETO-positive AML are associated with impaired event-free and overall survival. Blood,2006,107:1791-1799.
39. Thiede C, Koch S, Creutzig E, et al Prevalence and prognostic impact of NPM1 mutation in 1485 adult patients with acute myeloid leukemia(AML). Blood,2006, 107:4011-4020.
40.李磷,张悦,马晓瑭,等.原发性骨髓增生异常综合征患者NPM1基因突变的研究.中华血液学杂志,2010,31(12)：809-812.
41. Nguyen S, Leblanc T, Fenaux P, et al. A white blood cell index as the main prognostic factor in t(8;21) acute myeloid leukemia (AML):a survey of 161 cases from the French AML intergroup. Blood,2002,99:3517-23.
42. Pratz KW, Sato T, Murphy KM, et al. FLT3-mutant allelic burden and clinical status are predictive of response to FLT3 inhibitors in AML. Blood,2010,115:1425-1432.
43. Suzuki T, Kiyoi H, Ozeki K, et al. Clinical characteristics and prognostic implications of NPM1 mutations in acute myeloid leukemia. Blood,2005,106:2854-2861.
44. Boissel N, Renneville A, Biggio V, et al. for the Acute Leukemia French Association(ALFA). Prevalence, clinical profile, and prognosis of NPM mutations in AML with normal karyotype. Blood,2005,106:3618-3620.
45. Falini B, Nicoletti I, Martelli MF, et al. Acute myeloid leukemia carrying cytoplastic/mutated nucleophosmin(NPMc+AML):biologic and clinical features. Blood,2007,109:874-885.
46. Verhaak RG, Goudswaard CS, van Putten W, et al. Mutations in nucleophosmin(NPM1) in acute myeloid leukemia(AML):association with other gene abnormalities and previously established gene expression signatures and their favorable prognostic significance. Blood,2005,106:3747-3754.
47. Whitman SP, Archer KJ, Feng L, et al. Absence of the wild-type allele predicts poor prognosis in adult de novo acute myeloid leukemia with normal cytogenetics and the internal tandem duplication of FLT3:a cancer and leukemia group B study. Cancer Res,2001,61:7233-7239.
48. Gale RE, Green C, Allen C, et al. The impact of FLT3 internal tandem duplization mutant level, number, size, and interaction with NPM1 mutations in a large cohort of young adult patients with acute myeloid leukemia. Blood,2008,111:2776-2784.
49. Meshinchi S, Stirewalt DL, Alonzo TA, et al. Structural and numerical variation of FLT3/ITD in pediatric AML. Blood,2008,111:4930-4933.
50. Kottaridis PD, Gale RE, Frew ME, et al. The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia(AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials[J]. Blood,2001,98:1752-1759.
51. Raghavan M, Smith LL, Lillington DM, et al. Segmental uniparental disomy is a commonly acquired genetic event in relapsed acute myeloid leukemia. Blood, 2008,112:814-821.
52. Raghavan M, Lillington DM, Skoulakis S, et al. Genoem-wide single nucleotide polymorphism analysis reveals frequent partial uniparental dissomy due to somatic recombination in acute myeloid leukemias. Cancer Res,2005,65:375-378.
53. Kiyoi H, Towatari M, Yokota S, et al. Internal tandem duplication of the FLT3 gene is a novel modality of elongation mutation which causes constitutive activation of the product. Leukemia,1998,12:1333-1337.
54. Griffith J, Black J, Faerman C, et al. The structural basis for antoinhibition of FLT3 by the juxtamembrane domain. Mol Cell,2004,13:169-178.
55. Kern W, Schoch C, Hiddemann W. Prognostic significance of cytogenetics in relapsed acute myeloid leukaemia. Br J Haematol,2000,109:671-672.
56. Kern W, Haferlach C, Haferlach T, et al. Monitoring of minimal residual disease in acute myeloid leukemia. Cancer,2008,112(1):4-16.
1. Mrozek K, Heerema NA, Bloomfield CD. Cytogenetics in acute leukemia. Blood reviews,2004,18:115-36.
2. Grisendi S, Mecucci C, Falini B, et al. Nucleophosmin and cancer[J]. Nat Rev Cancer, 2006,6(7):493-505.
3. Lin MJ, Wang XW. Nucleophosmin and human cancer[J]. Cancer Detect Prev, 2006,30(6):481-490.
4. Hingorani K, Szebeni A, Olson MO. Mapping the functionaldomains of nucleolar protein B23 [J]. J Biol Chem,2000,275(32):24451-24457.
5. Lindstrom MS, Zhang Y. Ribosomal protein S9 is a novel B23/NPM-binding protein required for normal cell proliferation. J Biol Chem,2008,283:15568-15576.
6. Falini B, Mecucci C, Tiacci E, et al Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N Engl J Med,2005,352:254-266.
7. Gale RE, Green C, Allen C, et al. The impact of FLT3 internal tandem duplication mutant level, number, size, and interaction with NPM1 mutations in a large cohort of young adult patients with acute myeloid leukemia. Blood,2008,111(5):2776-2784.
8. Gorello P, Cazzaniga G, Alberti F, et al Quantitative assessment of minimal residual disease in acute myeloid leukemia carrying nucleophosmin (NPM1)gene mutations Leukemia,2006,20:1103-1108.
9. Nakagawa M, Kameoka Y, Suzuki R. Nucleophosmin in acute myelogenous leukemia. N Engl J Med,2005,352:1819-1820.
10. Cuomo ME, Knebel A, Morrice N, et al. p53-Driven apoptosis limits centrosome amplification and genomic instability downstream of NPM1 phosphorylation. Nat Cell Biol,2008,10:723-730.
11. Yogev O, Saadon K, Anzi S, et al. DNA damage-dependent translocation of B23 and p19ARF is regulated by the Jun N-ter-minal kinase pathway. Cancer Res,2008,68: 1398-1406.
12. A picelli AJ, Maggi LB Jr, Hirbe AC, et al. A non-tumor suppressor role for basal p19ARF in maintaining nucleolar structure and function. Mol Cell Biol,2008,28: 1068-1080.
13. Bonetti P, Davoli T, Sironi C, et al. Nucleophosmin and its AML-associated mutant regulate c-Myc turnover through Fbw7 gamma. J Cell Biol,2008,182:19-26.
14. Roti g, Rosati R, Bonasso R, et al. Denaturing high-performance liquid chromatography:a valid approach for identifying NPM 1 mutations in acute leukemia. J Mol Diagn,2006,8:254-259.
15.邹积艳,朱平,刘红星等.白血病NPM1基因突变检测方法的临床适用性比较.中华检验医学杂志,2009,32(1)：35-39.
16. Szankasi P, Jama M, Bahler DW. A new DNA-based test for detection of nuclephosmin exon 12 mutations by capillary electrophoresis. Diagn,2008,10: 236-241.
17. Abdel Rahman H, Farrag SA, El-Attar IA. AML1/ETO fusion gene in de novo pediatric acute myeloid leukemia:clinical significant prognostic implications. J Egypt Natl Canc Inst,2007,19:39-47.
18. Falini B, Bolli N, Shan J, et al. Both carboxy-terminus NES motif and mutated tryphophan(s) are crucial for aberrant nuclear export of nucleophosmin leukemia mutants in NPMc+AML. Blood,2006,107(11):4514-4523.
19. Falini B, Martelli MP, Bolli N, et al. Immunohistochemistry predicts nucleophosmin (NPM) mutations in acute myeloid leukemia. Blood,2006,108(6):1999-2005.
20. Thiede C, Koch S, Creutzig E, et al Prevalence and prognostic impact of NPM1 mutation in 1485 adult patients with acute myeloid leukemia(AML). Blood,2006, 107:4011-4020.
21. Thiede C, Creutzig E, Reinhardt D, et al. Different types of NPM1 mutations in children and adults:evidence for an effect of patient age on the prevalence of the TCTG-tandem duplication in NPM 1-exon 12. Leukemia,2007,21:366-367.
22. Brown P, Mclntyre E, Rau R, et al. The incidence and clinical significance of nucleophosmin mutations in childhood AML. Blood,2007,110:979-980.
23. Renneville A, Roumier C, Biggio V, et al. Cooperating gene mutations in acute myeloid leukemia:a review of the literature. Leukemia,2008,22:915-931.
24. Caudil JS, Sternberg AJ, Li CY, et al.C-terminal nucleophosmin mutations are uncommom in chronic myeloid disorders. Br J Haematol,2006,133:638-641.
25. Zhang Y, Zhang M, Yang L, et al. NPM1 mutations in myelodysplastic syndromes and acute myeloid leukemia with normal karyotype. Leuk Res,2007,31:109-111.
26. Schlenk RF, Dohner K, Krauter J, et al. Mutations and Treatment Outcome in Cytogenetically Normal Acute Myeloid Leukemia. N Engl J Med,2008,358:1909-18.
27. Andreeff M, Ruvolo V, Gadgil S, et al. HOX expression patterns identify a common signature for favorable AML. Leukemia,2008,22(11):2041-2047.
28. Ruan GR, Li JL,Qin YZ, et al. Nuclephosmin mutations in Chinese adults with acute myelogenous leukemia. Ann Hematol,2009,88:159-166.
29. Yan LZ, Chen SN, Liang JY, et al. Analysis of NPM1 gene mutations in Chinese adults with acute myeloid leukemia. Int J Hematol,2007,86:143-146.
30. Schnittger S, Schoch C, Kem W, et al Nucleophosmin gene mutations are predictors of favorable prognosis in acute myelogenous leukemia with a normal karyotype Blood,2005,106:3733-3739.
31. Choudhary C, Schwable J, Brandts C, et al. AML-associated FLT3 kinase domain mutations show signal transduction differences compared with FLT3 ITD mutations. Blood,2005,106(1):265-273.
32. Koh Y, Park J, Bae EK, et al. Non-A type nucleophosmin 1 gene mutation predicts poor clinical outcome in de novo adult acute myeloid leukemia:differential clinical importance of NPM1 mutation according to subtype. Int J Hematol,2009,90(1):1-5.
33. Boissel N, Renneville A, Biggio V, et al. Prevalence,clinical profile, and prognosis of NPM mutations in AML with normal karyotype. Blood,2005,106(10):3618-3620.
34. Chou WC, Tang JL, Wu SJ, et al. Clinical implications of minimal residual disease monitoring by quantitative polymerase chain reaction in acute myeloid leukemia patients bearing nucleophosmin(NPM1)mutations. Leukemia,2007,21(5):998-1004.
35. Rosnet O, Marchetto S, Delapeyriere O, et al. Murine FLT3, a gene encoding a novel tyrosine kinase receptor of the PDGFR/CSF1R family. Oncogene,1991,6: 1641-1650.
36. Matthews W, Jordan CT, Wiegand GW, et al. A receptor tyrosine kinase specific to hematopoietic stem and progenitor cell enriched populations. Cell,1991,65: 1143-1152.
37. Rosnet O, Schiff C, Pebusque MJ, et al. Human FLT3/FLK2 gene:cDNA coloning and expression in hematopoietic cells. Blood,1993,82:1110-1119.
38. Rosmarin AG, Yang Z, Resendes KK. Transcriptional regulation in myelopoiesis: hematopoietic fate choice, myeloid differentiation, and leukemogensis. Exp Hematol, 2005,33:131-143.
39. Griffith J, Back J, Faerman C, et al. The structural basis for auto inhibition of FLT3 by the juxtamembrane domain [J]. Mol Cell,2004,30:169-178.
40. Stirewalt DL, Radich JP. The role of FLT3 in hematopoietic maglignancies [J]. Nat Rev Cancer,2003,3(9):650-665.
41. Schenone S, Brullo C, Botta M. Small molecular ATP-competitive inhibitors of FLT3: a chemical overview [J]. Curr Med Chem,2008,15(29):3113-3132.
42. Nakao M, Yakota S, Iwai T, et al. Internal tandem duplication of the flt3 gene found in acute myeloid leukemia. Leukemia,1996,10:1911-1918.
43. Kelly LM, Liu Q, Kutok JL, et al. FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myeloproliferative disease in a murine bone marrow transplant model. Blood,2002,99:310-318.
44. Abu-Duhier FM, Goodeve AC, Wilson GA, et al. Genomic structure of human FLT3: implications for mutational analysis. Br J hematol,2001,113(4):1076-77.
45. Chunaram Choudhary, Joachim Schwable, et al. AML-associated Flt3 kinase domain mutations show signal transduction differences compared with Flt3 ITD mutations [J]. Blood,2005,106:265-273.
46. Frohling S, Schlenk RF, Breitruck J, et al. Prognostic significance of activating FLT3 mutations in younger adults(16 to 60 years) with acute myeloid leukemia and normal cytogenetics:a study of the AML Study Group Ulm. Blood,2002,100(13): 4372-4380.
47. Schnittger S, Schoch C, Dugas M, et al. Analysis of FLT3 length mutations in 1003 patients with acute myeloid leukemia:correlation to cytogenentics, FAB subtype, and prognosis in the AML CG study and usefulness as a marker for the detection of minimal residual disease. Blood,2002,100(1):59-66.
48. Shih LY, Huang CF, et al. Heterogeneous patterns of Flt3 ASP835 mutations in relapsed de novo acute myeloid leukemia:a comparative analysis of 120 paired diagnostic and relapse bone marrow samples [J]. Clinical Cance Research, 2004,10:1326-1332.
49. Rebekka Grundler, Christian Thiede, et al. Senxitivity toward tyrosine kinase inhibitors varies between different activating mutations of the FLT3 receptor [J]. Blood,2003,102:646-651.
50.黄晶,刘力华,张玲等PCR-SSCP检测急性髓系白血病患者FLT3基因及FLT3/ITD基因突变.中国实验诊断学,2009,13(8)：1090-1092.
51. Vaughn CP, Elenitoba Johnson KS. High-resolution melting analysis for detection of internal tandem duplications [J]. J Mol Diagn,2004,6:211-216.
52. Shih L Y, Lin TL, Wang PN, et al. Internal tandem duplication of fms-like tyrosine kinase 3 is associated with poor outcome in patient with myelodysplastic syndrome. Cancer,2004,101(5):989-998.
53. Bench AJ, Erber WN, Scott MA. Molecular genetic analysis of haematological malignancies:I. Acute leukemias and myeloproliferative disorders [J]. Clin Lab Haematol,2005,27:148-171.
54. Kottardis PD, Gale RE, Linch DC. Flt3 mutations and leukemia. Br J Haematol, 2003,122(4):523-538.
55. Whitman SP, Maharry K, Radmacher MD, et al. FLT3 internal tandem duplication associates with adverse outcome and gene and microRNA-expression signatures in patients 60 years of age or older with primary cytogenetically normal acute myeloid leukemia:a Cancer and Leukemia Group B study. Blood,2010,116(18):3622-3626.
56. Zwaan CM, Meshinchi S, Radich JP, et al. FLT3 internal tandem duplication in 234 children with acute myeloid leukemia:prognostic significance and relation to cellular drug resistance. Blood,2003,102(7):2387-2394.
57. Gale RE, Hills R, Kottardis PD, et al.No evidence that FLT3 status should be considered as an indicator for transplantation in acute myeloid leukemia(AML):an analysis of 1135 patients, excluding acute promyelocytic leukemia, from the UK MRC AML 10 and 12 trials. Blood,2005,106:3658-3665.
58. Stirewalt DL, Kopechy KJ, Meshinchi S, et al. Size of flt3 intenal tandem duplications has prognostic significance in patients with acute myeloid leukemia. Blood,2006,107:3724-3726.
59. Meshinchi S, Stirewalt DL, Alonzo TA, et al. Structural and numerical variation of FLT3/ITD in pediatric AML. Blood,2008,111:4930-4933.
60. Whitman SP, Archer KJ, Feng L, et al. Absence of the wild-type allele predicts poor prognosis in adult de novo acute myeloid leukemia with normal cytogenetics and the internal tandem duplization of FLT3:a cancer and leukemia group B study. Cancer Res,2001,61(19):7233-7239.
61. Thiede C, Steudel C, Mohr B, et al. Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia:association with FAB subtypes and identification of subgroups with poor prognosis. Blood,2002,99(12):4326-4335.
62. Baldus CD, Thiede C, Soucek S, et al. BAALC expression and FLT3 internal tandem duplication mutations in acute myeloid leukemia patients with normal cytogenetics prognostic implications. J Clin Oncol,2006,24(5):790-797.
63. Kottaridis PD, Gale RE, Frew ME, et al. The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia(AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials[J]. Blood,2001,98:1752-1759.
64. Kayser S, Schlenk RF, Londono MC, et al. Insertion of FLT3 internal tandem duplication in the tyrosine kinase domain-1 is associated with resistance to chemotherapy and inferior outcome. Blood,2009,114(12):2386-2392.
65. Yamamoto Y, Kiyoi H, Nakano Y, et al. Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies. Blood,2001,97(8): 2434-2439.
66. Levis M. Recent advances in the development of small-molecule inhibitors for the treatment of acute myeloid leukemia. Current opinion in Hematology,2005,12(1): 55-61.
67. Pratz KW, Sato T, Murphy KM, et al. FLT3-mutant allelic burden and clinical status are predictive of response to FLT3 inhibitors in AML. Blood,2010.115:1425-1432.
68. Breitenbuecher F, Markova B, Kasper S, et al. A novel molecular mechanism of primary resistence to FLT3-kinase inhibitors in AML. Blood,2009,113(17): 4063-4073.
69. Von Bubnoff N, Engh RA, Aberg E, et al. FMS-like tyrosine kinase 3-internal tandem duplication tyrosine kinase inhibitors display a nonoverlapping profile of resistance mutations in vitro. Cancer Res,2009,69(7):3032-3041.
70. Zhou J, Bi C, Janakakumara JV, et al. Enhanced activation of STAT pathways and overexpression of surviving confer resistance to FLT3 inhibitors and could be therapeutic targets in AML. Blood,2009; 113(17):4052-4062.
71. Stolzel F, Steudel C, Oelschlagel U, et al. Mechanisms of resistance against PKC412 in resistant FLT3-ITD positive human acute myeloid leukemia cells. Ann Hematol, 2010,89(7):653-662.
72. Yao Q, Nishiuchi R, Li Q, et al. FLT3 expressing Leukemias are selectively sensitive to inhibitors of the molecular chaperone heat shock protein 90 through destabilization of signal transduction-associated kinases. Clin Cancer Res,2003,9:4483-4493.
73. Mohi MG, Boulton C, Gu TL, et al. Combination of rapamycin and protein tyrosine kinase (PTK) inhibitors for the treatment of leukemias caused by oncogenic PTKs. Proc Natl Acad Sci USA,2004,101:3130-3135.
74. Al Shaer L, Walsby E, Gilkes A, et al. Heat shock protein 90 inhibition is cytotoxic to primary AML cells expressing mutant FLT3 and results in altered downstream signaling. Br J Haematol,2008,141 (4):483-493.
75. Nishioka C, Ikezoe T, Yang J, et al. MS-275, a novel histone deacetylase inhibitor with selectivity against HDAC1, induces degradation of FLT3 via inhibition of chaperone function of heat shock protein 90 in AML cells. Leuk Res, 2008,32(9):1382-1392.
76. Piloto O, Nguyen B, Huso D, et al. IMC-EB10, an anti-FLT3 monoclonal antibody, prolongs survival and reduces nonobese diabetic/severe combined immunodeficient engraftment of some acute lymphoblastic leukemia cell lines and primary leukemic samples. Cancer Res,2006,66(9):4843-4851.
77. Zeng Z, Shi YX, Samudio IJ, et al. Targeting the leukemia microenvironment by CXCR4 inhibition overcomes resistance to kinase inhibitors and chemotherapy in AML. Blood,2009,113(24):6215-6224.
